DESCRIPTION: (Provided by the applicant) RUSH proteins are SWI/SNF-related transcription factors with RING-finger signatures near their Ctermini. Long suspected of mediating protein-protein interactions, the RING motif was used to clone a binding partner. The RING-finger binding protein (RFBP) is a Type IV P-type ATPase embedded in the inner nuclear membrane. This putative phospholipid pump has conserved sequences for two loop segments, an ATP-binding site, a phosphorylation domain and transmembrane passes potentially involved in substrate binding and translocation. However, RFBP differs from authentic Type IV P-type ATPases in several important ways. It has only three of four highly conserved NH2-terminal transmembrane passes. Topographically the orientation of the adjacent hydrophilic domains and the determinants of transport specificity are altered suggesting RFBP is not a transport protein. The small, hydrophilic loop extends into the perinuclear space that is contiguous with the lumen of the endoplasmic reticulum. The large, conformationally flexible loop extends into the nucleoplasm to contact euchromatin. Competitive reverse-transcriptase polymerase chain reaction and HPLC analysis showed that endometrial RFBP mRNA expression is hormonally regulated. The goal to more completely characterize RFBP will be achieved with three aims. In aim one, a 3-4-kb fragment of 5'-flanking sequence will be subjected to functional mapping to identify positive as well as negative attenuating elements. Deletion analysis and mutagenesis will help identify combinatorial interactions that govern RFBP transcription. In aim two, subregions of the RING motif and RFBP responsible for protein-protein interactions will be identified with glutathione 5-transferase-pulidown assays and yeast two-hybrid screens. Mutational analysis will be used to map the minimal protein-binding domains. In aim three, the effects of hormones on the dynamic association of RFBP with active chromatin, matrix associated splicing complexes and RING-finger containing factors will be determined by laser scanning confocal microscopy and Western analysis. The physical association of a hormone-dependent RING-finger binding protein with transcriptionally active chromatin suggests that RFBP plays a role in the subnuciear trafficking of transcription factors with RING motifs. The ultimate goal of understanding the regulatory role of hormones in the nuclear partitioning of transcription factors is central to defining the mechanism of hormone action in the uterus, and may be critical to good reproductive health.